The circumstances that predispose to development of HIV and SIV encephalitis (SIVE) are not known; current evidence underscores the importance of the CNS macrophage. The role of CNS macrophages as a target of HIV and SIV infection, and their contribution to the development of CNS lesions is well established. However the identification of distinct monocyte/macrophage subpopulations that carry virus to the CNS and macrophage subsets within the CNS that are the target for infection is not resolved. We have used immunohistochemistry to define phenotypic differences between the resident parenchymal microglia, perivascular macrophages, and multinucleated giant cells. All of these cells express antigens of myeloid lineage including complement receptor 3 (CD11b), CD4, and CD68. Perivascular macrophages and multinucleated giant cells (MNGC) but not parenchymal microglia expressed LPS receptor (CD14) and LCA (CD45). Perivascular macrophages and parenchymal microglia upregulate or express de novo CD11b, CD40, and B7-2 (CD86) with higher level of expression of these antigens within the subcortical white matter. In several cases we observed that macrophages making up MNGC had either lost or down regulated CD4. Using this immunophenotype pattern to differentiate between brain macrophages and either double staining for viral protein or in situ hybridization for viral nucleic acid, we found that a population of perivascular brain macrophages is the major cell infected in the CNS of animals with SIVE. We further demonstrate based upon immunohistochemistry for CD14 and CD45 that the MNGC are derived from a population of perivascular macrophages and not the parenchymal microglia. These studies demonstrate the ability to differentiate between subpopulations of brain macrophages underscoring the primary role of the perivascular macrophage as an early and major target of SIV.